Francisella tularensis is a gram-negative bacterium and the causative agent of tularemia, a potentially deadly disease. It is a threat as an agent of bioterrorism because of the highly infectious nature of the airborne organism. There is not currently an approved vaccine to protect against this agent. In the mammalian host, the bacterium is an intracellular pathogen of macrophages and other cells;they reside within membrane-bound phagosomes early in infection and subsequently escape into the cytoplasm where they replicate. Iron acquisition systems in the iron-scarce host environment are critical virulence determinants in many pathogens. Although iron is important for proliferation of F. tularensis within macrophages, iron acquisition mechanisms in this organism are uncharacterized. This proposal is based on the hypothesis that iron acquisition is a key element in pathogenesis in the mammalian host. Many microorganisms secrete small molecules called siderophores to chelate iron from a variety of sources. We have shown that the F. tularensis bacteria secrete a siderophore whose production is dependent on the fsIA gene. Iron-siderophore uptake by gram-negative bacteria is universally dependent on the TonB-ExbB-ExbD complex, but the F. tularensis genome does not encode homologs of these proteins. We have determined that the fslE gene is necessary for iron acquisition under conditions of iron limitation and from host sources. The goal of this proposal is to investigate the roles of the fslABCDEF genes in iron uptake in F. tularensis. Specific aim 1 of this project is the phenotypic analysis of deletion mutants in each of the fsl genes. We will test for siderophore expression and iron uptake, and we will also assess the virulence of the mutants in a mouse model of infection. Specific aim 2 is the characterization of the Fsl gene products. We will determine the subcellular localization of each of the gen products using biochemical and immunological techniques. We will characterize the activity of the putative enzymes FsIA and FslC. We will test the ability to reconstitute the iron acquisition system involving FslE and/or FsIF in Escherichia coli as a model system to study a novel paradigm for iron uptake. Understanding the mechanisms to acquire iron will enable us to harness them in therapeutic applications and in development of defined subunit vaccine and vaccine strains so that we may be better prepared to deal with a potential tularemic outbreak.